RNA interference (RNAi) is an evolutionarily conserved cellular defense against foreign or parasitic genetic elements. This regulatory system ensures genetic stability. Loss of RNAi in the soma may lead to cancer;loss of RNAi in the germ line may cause birth defects. In Drosophila melanogaster, the proteins Dicer-2 and R2D2 are essential for the RNA interference (RNAi) pathway. These proteins form a stable heterodimer in vitro, and without Dicer-2, R2D2 is unstable in vivo. Yet in vitro analysis indicates that Dicer-2 can carry out one of its functions-cleaving dsRNA-in the absence of R2D2. The goal of this study is to identify the amino acids required for the binding of these two proteins to each other, so as to design mutant flies in which I can test whether R2D2 must be bound to Dicer-2 during dicing or, rather, must only be present at subsequent steps in the production of an active RNAi enzyme complex. To this end, I propose to use a reverse yeast two- hybrid screen to select mutants of Dicer-2 that are unable to bind R2D2. I will carry out quantitative biochemical tests to identify a mutant that retains its ability to cleave long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs), the guides that direct RNAi. I will generate transgenic flies that have two forms of Dicer-2: the mutant form of Dicer-2 that no longer binds R2D2 but is able to cleave dsRNA, as well as an existing mutant Dicer-2 that binds R2D2 but is diminished in its ability to cleave dsRNA. Using established techniques, I will examine these flies to determine whether RNAi can occur in the living fly when the processing dsRNA into siRNAs and the loading of the siRNAs into functional complexes are decoupled. I will also use molecular tools, including quantitative RT-PCR and tiling microarrays, to monitor the levels of genetic elements that are normally silenced, and high throughput sequencing of the small RNA populations in these flies to determine the in vivo consequences of separating these processes. PUBLIC HEALTH RELEVANCE: A better understanding of the biochemical basis for RNA interference (RNAi) will lead to better experimental RNAi tools. Such tools provide the underpinnings of modern somatic genetics and have become an essential part of academic and pharmaceutical discovery. This work will lead to the development of improved therapeutics for diseases that have a genetic basis, including some cancers, Huntington's disease, and Parkinson's disease.